Ophthmalic irrigating solution

ABSTRACT

An ophthalmic irrigating solution useful for irrigating the human eye contains sodium, potassium, magnesium, calcium, chloride, and bicarbonate ions as well as dextrose and glutathione in proportions consistent with the osmotic stability and continued metabolism of the endothelial cells. The irrigating solution is prepared by mixing a first basic solution which provides the bicarbonate and a second acidic solution which provides the calcium, magnesium, dextrose and glutathione. The first and second solutions may be stored as stable, sterile solutions for extended periods of time and mixed within 24 hours of use.

The present invention relates to electrolyte solutions for use withinthe human body and more particularly to solutions useful for irrigatingthe eye during surgery.

Any scission into the human body is detrimental to the human body andinvariably results in cell loss. The need to keep cell loss to a minimumis particularly crucial during any surgical procedure performed ondelicate and irreplaceable tissues such as the tissues of the eye.

The cornea of the eye is comprised of five layers: epithelium, Bowman'smembrane, stroma, Decemet's membrane, and endothelium. The endotheliumlayer is particularly vulnerable to trauma as the endothelial cells areinfrequently, if ever, replaced as a normal process in the adult life.The endothelium is principally responsible for the maintenance of theproper state of hydration of the stromal layer. The stromal layer has atendency to imbibe fluid, a tendency which is counterbalanced by outwardfluid transport via the endothelium. If the proper fluid balance is notmaintained in the stromal layer, the cornea thickens and thecharacteristic transparency of the cornea is lost. Accordingly, cellloss or damage in the endothelial layer will result in decreased vision.Failure of the endothelium to perform its fluid transport function forshort periods of time will result in corneal thickening and visualclouding. Because of the importance of, and the vulnerability of theendothelial layer, it is necessary during eye surgery, such as cataractsurgery or corneal transplants, to make provisions for the protection ofthe endothelial cells.

A significant factor causing cell loss during tissue scission is thetraumatic change in environment experienced by the internal cells.Exposure to the atmosphere presents a far different environment for thecells than is provided by the natural fluids in which they are bathed.To simulate the natural cellular environment and thereby prevent celldamage, exposed tissue during surgery is frequently irrigated insolutions which attempt to approximate natural body fluids. The value ofbathing eye tissue during surgery to prevent cell damage has long beenrecognized. For internal ocular tissues such as the endothelium, theaqueous humor is the natural bathing fluid and, hence, an ophthalmicirrigating solution to protect the endothelium should as closely aspossible resemble the aqueous humor.

Of primary concern in a tissue irrigating solution is that theosmolality of the solution be generally isotonic with cellular fluids soas to maintain equal osmotic pressure within and without the cellmembranes. To this end, one of the early ophthalmic irrigating solutionswas isotonic (0.9%) saline. However, as has long been recognized,isotonic saline is quite inadequate as an ophthalmic irrigating solutionand has been shown to result in endothelial cell swelling, cell damage,and consequent corneal clouding.

Because of the inadequacy of isotonic saline, various alternativeelectrolyte solutions have been proposed as ophthalmic irrigatingsolutions in attempts to provide solutions which more closely resemblethe aqueous humor and prevent cell damage and corneal clouding. Standardelectrolyte solutions, primarily intended for injection solutions suchas Ringer's solution, and lactated Ringer's solution have been used asophthalmic irrigating solutions because of their wide availability assterile solutions.

A solution intended for ophthalmic irrigation known as balanced saltsolution (BSS) has also been developed. BSS contains the essential ions,calcium, sodium, potassium, magnesium and chloride in generally optimalconcentrations for ocular tissue, and has an acetate-citrate buffersystem which is compatible with divalent calcium and magnesium ions.

The various electrolyte solutions used for ophthalmic irrigation havebeen improvements over normal saline by providing necessary ions inaddition to Na⁺ and Cl⁻ as provided by isotonic saline. Mg⁺⁺ is animportant cofactor for adenosine triphosphatase, an enzyme which playsan important role in mediating the fluid transport pump in the eye. Ca⁺⁺is necessary to maintain the endothelial junction. K⁺ is an importantfactor in many biochemical processes, and the fluid transport pump ofthe endothelium requires a proper Na⁺ /K⁺ ratio. The previously knownelectrolyte solutions used to irrigate ocular tissue have reduced butnot eliminated corneal swelling and cell damage.

The need for improved ophthalmic irrigating solutions continues,particularly in view of new surgical techniques which may probe deeperinto the eye and require several hours of operating time. Surgicaladvances now permit surgery in the vitreous (posterior) chamber toremove opacified vitreous humor or to repair retinal detachment. Suchoperations require significant time, e.g., 1 to 3 hours, and largevolumes of irrigating solution, e.g., 100-1000 ml.

During eye surgery and particularly during surgery which requiresextended periods of time, proper electrolytic balance alone isinsufficient to retain normal corneal thickness. To maintain propercorneal thickness and prevent cell damage, an irrigating solution, inaddition to electrolytic balance, must provide metabolic support andmust particularly provide factors needed for the enzyme-mediated Na⁺ /K⁺pump system through which excess fluid is removed from the stroma.

To incorporate factors necessary for sustained metabolism by endothelialcells, glutathione-bicarbonate-Ringer's solution (GBR) was developed inwhich NaHCO₃, glutathione, dextrose and adenosine (an optionalingredient) are added to Ringer's solution. Bicarbonate, dextrose andglutathione have been shown to be important factors in maintainingstructural integrity of endothelial cells. The aqueous humor has abicarbonate buffer system. Dextrose (d-glucose) provides a substrate forvarious metabolic pathways, and glutathione has been shown to aid themetabolic pump mechanism by maintaining proper Na⁺ /K⁺adenosine-triphosphotase. GBR has been shown to be effective inmaintaining corneal thickness and endothelial cell integrity for up tothree hours.

While the effectiveness of GBR ocular irrigating solution has been shownboth in vivo and in vitro, its use in surgery has been limited forreasons of stability and sterility. It is to be appreciated thatsterility of an ophthalmic irrigating solution is absolutely essential.To insure sterility, it is desirable that an irrigating solution beprepackaged so that the quality and sterility may be closely monitoredand tested as contrasted with an extemporaneously mixed solution asmight be prepared in a hospital pharmacy. The solution will perfuse theeye in essentially a closed system where even a small number oforganisms could produce an overwhelming endophthalmitis, as pseudomonasis one of the very few organisms that has very few metabolicrequirements and can grow with a minimal nutrient supply, such asphosphate and bicarbonate. Dr. Jan Worst has reported on a series ofinfections in Europe with pseudomonas-contaminated irrigating solutions.(January 1978, American Intraocular Implant Society Journal).

GBR may not be prepackaged due to the long term incompatability and/orinstability of its various moieties. Of the moieties added to Ringer'ssolution to formulate GBR, bicarbonate is perhaps the most important(McEnerney et al. Investigative Ophthalmology and Visual Science, Vol.16 No. 7, July 1977). Unfortunately, the bicarbonate as well as thephosphate in a bicarbonate-phosphate buffer system form insolubleprecipitates with Mg⁺⁺ and Ca⁺⁺. While at the ionic concentrationsuseful in ophthalmic irrigation, precipitation is not a problem infreshly prepared solution, long term storage is proscribed. As insolublecrystals introduced into the eye will cloud vision, the importance ofkeeping an ocular irrigating solution free of insoluble precipitates maybe readily appreciated. The fortification of Ringer's injection withsodium bicarbonate and dextrose and used with an Ocutome® Fragmatome™instrument deposits pure crystals of calcium bicarbonate in theinstrument system as well as inside the eye on the retina, vitreousdisk, iris and on exposed uvea or sclera. (Dr. Connor O'Malley, "SaltContamination of the Eye - An Infusion Hazard". Ocutome/FragmatomeNewsletter, Vol. 4, No. 4, 1979).

Complicating the maintenance of GBR's stability is the fact that the pHof GBR will gradually increase due to the inadequacy of thebiocarbonate-phosphate buffer. To provide proper pH, i.e., about 7.4,the pH must be monitored and adjusted with CO₂ immediately prior to useand even during use. The chances for contamination during pH adjustmentare great.

A further factor which proscribes long-term storage of GBR is theunavailability of a proper pH at which all of the moieties are stable.Several moieties of GBR are unstable at the physiological pH of about7.4. Below a pH of about 8, bicarbonate generally decomposes to CO₂,resulting both in a loss of bicarbonate concentration and increased pH.On the other hand, glucose stability requires a pH of less than about 6.Glutathione, while biologically effective either in reduced or oxidizedform, is preferred in the oxidized form because the reduced form quicklyoxidizes in aqueous solutions preventing proper labeling of theirrigating solution. Oxidized glutathione (glutathione disulfide) isunstable over extended periods of time at a pH of above about 5. Becauseof the demonstrated efficacy of GBR as an ocular irrigating solution, itwould be desirable to provide a formulation which contains the essentialfactors found in GBR and which may be stored in a sterilized form foruse in eye surgery.

Accordingly, it is a primary object of the invention to provide a stablesterile ophthalmic irrigating solution which, in addition to correctelectrolyte balance, provides factors necessary for continued metabolismin the endothelial cells, maintenance of the fluid transport pumpsystem, and consequential maintenance of proper corneal thickness andclarity.

The present invention is directed generally to a two-part solutionsystem which includes a basic solution and an acidic solution. Thecomposition and concentration of the two solutions are such that theyare individually stable and may be separately stored for long periods.When mixed together, the two solutions form a solution which containsthe necessary factors to maintain endothelial cell integrity and cornealthickness during ocular surgery. The combined ophthalmic irrigatingsolution contains the necessary ions, Ca⁺⁺, Mg⁺⁺, Na⁺, K⁺ and Cl⁻ in abicarbonate-phosphate buffer as well as oxidized glutathione anddextrose. (As used herein, "glutathione" is used to refer to either theoxidized form of glutathione (GSSG) or the reduced form (GSH).Irrespective of the form used to prepare the solution, the glutathionewill be in its oxidized form in the final ocular solution to avoidconfusion in labeling, and the solutions will generally be prepared withoxidized glutathione.) The solution may also contain adenosine.

The electrolytes are provided in proportions conducive to cellularintegrity and continued cell metabolism. Preferably, the proportions ofelectrolytes resemble BSS, a solution particularly formulated forophthalmic irrigation rather than Ringer's solution, which wasformulated for injection into the cardiovascular system. Preferably, theirrigating solution is more substantially buffered than GBR so that thepH does not continually change as does the pH of GBR. However, it isintended that the scope of the present invention be limited only toproportions of electrolytes compatible with ocular tissue.

The ophthalmic irrigating solution contains from about 130 to about 180mM/l Na⁺, from about 3 to about 10 mM/l K³⁰ , from about 1 to about 5mM/l Ca⁺⁺, from about 0.5 to about 4 mM/l Mg⁺⁺, and from about 130 toabout 210 mM/l Cl⁻. To maintain osmotic stability of the cells, theosmolality is between about 250 and about 350 mOsm and preferably about290-320. So as to closely match the physiological pH of 7.4, the pH ofthe final ophthalmic irrigating solution is between about 6.8 and about8.0 preferably about 7.2-7.8. To approximate the aqueous humor andmaintain the fluid pump system, the bicarbonate concentration in thecombined ophthalmic irrigating solution is between about 10 and about 50mM/l. To stabilize the pH, a further buffering agent is provided.Preferably, the buffering agent is phosphate which is provided insufficient quantity so that the final phosphate concentration of theirrigating solution is between about 1 and about 5 mM/l. The finalirrigating solution contains between about 2 and about 10 mM/l glucoseand between 0.03 and about 0.5 mM/l of oxidized glutathione or theequivalent amount of reduced glutathione. (One mole of oxidizedglutathione is the equivalent of two moles of reduced glutathione.)

The basic solution provides the phosphate and bicarbonate bufferingmoieties, preferably in the form of dibasic sodium phosphate and sodiumbicarbonate. The pH of the basic solution is adjusted close to thephysiological pH of 7.4, preferably to between about 7.2 and about 7.8.As hereinbelow mentioned, the pH of the bicarbonate-containing solutionis preferably above about 8.0 to prevent decomposition of thebicarbonate. It has been found, however, that the bicarbonate may bestabilized if it is added to a solution with a pH of above about 8 andthereafter adjusted to a pH between 7 and 8. Accordingly, when the basicsolution is prepared, Na₂ HPO₄ is added prior to the addition of NaHCO₃so that NaHCO₃ is dissolved in a solution with a pH of between about 8and about 9. The solution is thereafter adjusted with dilute acid, suchas H₂ SO₄, H₃ PO₄ or HCl, to the desired final pH of the basic solution.Alternatively, carbon dioxide may be added to adjust the pH.

Potassium and additional sodium are provided in the basic solution inthe form of sodium and potassium salts, such as sodium or potassiumchlorides, sulfates, acetates, citrates, lactates, and gluconates. Thesodium and potassium are compatible with all of the moieties present inthe finished ophthalmic irrigating solution, and sodium chloride andpotassium chloride may be added to either solution. However, in view ofthe fact that the basic solution provides the buffer system, the pH ofthe final ophthalmic solution may be more accurately determined if allcompatible salts are included in the basic solution.

The acidic solution provides the Ca⁺⁺ in the form of calcium chloride,the Mg⁺⁺ in the form of magnesium chloride, the glutathione and thedextrose. The pH is adjusted to below about 5 to provide long-termstability to the dextrose and oxidized glutathione.

Because of the requirement that the acidic solution have a low pH, it ispreferable that the volume of the basic solution greatly exceed thevolume of the acidic solution and that the acidic solution contain nobuffering agents. The acidic solution may be adjusted below a pH ofabout 5 with a relatively small amount of HCl. Because the acidicsolution is unbuffered, its pH is a reflection of the acid concentrationand less acid is needed to adjust the pH of a small volume. The largevolume of buffered basic solution may be adjusted very close to thefinal pH of the irrigating solution and will be relatively unaffected bythe addition of the small volume of the acidic solution. Preferably, theratio of the basic solution volume to the acidic solution volume isabout 10 to 1 to about 40 to 1.

The basic solution and the acidic solution are sterilized and separatelybottled or contained under sterile conditions by standard techniques,such as autoclaving or use of sterilizing filters. To avoid the need formeasuring volumes in the hospital which may introduce possible errorand/or contamination, it is highly preferred that particular volumes ofthe basic and acidic solutions be bottled so that adding the entirecontent of a container of the acidic solution to the entire content of acontainer of the basic solution results in the correctly formulatedophthalmic irrigating solution. The solutions may be mixed up to 24hours before a surgical procedure without the occurrence of significantpH change and without the formation of detectable precipitates andwithout degradation.

Precautions to maintain sterility of the solutions and to insure correctmixing of the acidic and basic solutions cannot be overdone. While themanufacturer may take all due precautions to maintain quality control,carelessness by a technician may render all such precautions for naught.Any opening of a container, no matter how carefully performed, increasesthe likelihood of contamination in the contents. As one method ofsubstantially eliminating the possibility of improper mixing and toreduce the likelihood of contamination, the solutions may be shipped ina container having a first chamber for the basic solution, an isolatedsecond chamber for the acidic solution and means to communicate thechambers without opening the container. The use of such containers areknown for the shipment of multi-part medical solutions. As one example,a container may have a lower chamber containing a measured volume of thebasic solution separated by a membrane from an upper chamber containinga measured volume of the acidic solution. The container cap may includea plunger means which, when depressed, causes a sharp point or bladedepending therefrom to break the membrane. The container is thereafteragitated, as by shaking, to complete the sterile mixing in proper volumeof the acidic and basic solutions.

The proper mixing of the acidic and basic solutions may also be carriedout by aseptically removing the acidic solution from its package with asterile syringe and needle and aseptically adding to the contents of thebasic solution package through the rubber stopper. Alternately, asterile double-ended needle can be used to transfer the acidic solutionto the basic solution by aseptically inserting one end of the needleinto the vial containing the acidic solution and then asepticallyinserting the other end of the needle into the basic solution, packagewhereby the vacuum maintained therein transfers the acidic solution tothe basic solution and is mixed.

The manner of providing an ocular irrigating solution which is sterile,storable, and which provides the correct electrolyte balance as well asfactors necessary for cell metabolism may now be fully appreciated. Bysegregating the phosphate and bicarbonate ions from the divalentmagnesium and calcium ions, the buildup of insoluble precipitates iseliminated. The solution which contains the bicarbonate is slightlybasic and the bicarbonate therein is stabilized as a result of initiallydissolving the bicarbonate at a sufficiently basic pH. The othersolution is sufficiently acidic to stabilize dextrose and oxidizedglutathione. The basic and acidic sterile solutions are simply combinedto provide the correctly formulated irrigating solution without addingfurther substances, adjusting the pH or introducing apparatus, any ofwhich are potentially contaminating. While the final combined irrigatingsolution is not stable for an extended period of time, it is stable forsufficiently long periods, i.e., at least 24 hours so that theirrigating solution may be carefully mixed under unhurried conditionsprior to an operation and remain stable throughout the operation. Thebuffer system is adequate to maintain a generally constant pH for atleast a 24-hour period eliminating the need to monitor or adjust the pH.

EXAMPLE

Separate sterile basic and acidic solutions were made and packaged. Asample of the basic solution and a sample of the acidic solution weremixed, and the combined solution was tested for stability as well as theability of maintaining the structural integrity and function of rabbitand human endothelia during in vitro perfusion, i.e., simulatingintraocular irrigation.

Part I (Basic Solution) was made by dissolving 2.582 Kg sodium chloride,138.2 grams potassium chloride, and 151.55 grams anhydrous dibasicsodium phosphate in water for injection at about 20° C. Then 918.75grams of sodium bicarbonate were added and dissolved. Additional waterfor injection was added to make about 350 Kg batch weight and 1 N HCladded (about 1 liter) to adjust pH to about 7.4. The solution was thenpassed through a 0.45 micron Millipore filter and each bottle (USP TypeI glass) was filled with about 480 ml of solution. The filled bottleswere then stoppered, vacuumed and sealed. The sealed bottles weresterilized by autoclaving at 121° C. for about 23 minutes.

Part II (Acidic Solution) was made by dissolving 385 grams calciumchloride dihydrate, 500 grams magnesium chloride hexahydrate, 2300 gramsdextrose, and 516 grams of 94% oxidized glutathione (glutathionedisulfide) in water for injection to make a final batch volume of about100 liters. The solution was then sterile filtered through a 0.22 micronmembrane filter and aseptically filled into presterilized 20 ml Type Iglass vials and sealed with presterilized rubber stoppers. (Alternately,the solution can be packaged in Type I glass ampules.)

After adding 20 ml of Part II (acidic solution) to a 480 ml bottle ofPart I (basic solution) and mixing, in vitro corneal endothelialperfusion studies were carried out on both rabbit and human donorcorneas along with commercial lactated Ringer's solution and Plasmalyte148 (pH 7.4) for comparison. Corneas from rabbits and human donorcorneas were excised and mounted in a dual chambered specularmicroscope. A swelling rate (μ/hr) was calculated by regression analysisfrom measurements of sequential changes in corneal thickness. Isolatedcorneas were perfused at 37° C. at 15 mm Hg with each of the testsolutions for up to three hours. Each experiment was studied as a pair,one cornea receiving the ophthalmic irrigating solution of the instantinvention and the other cornea perfused with either lactated Ringer'ssolution or Plasmalyte. The corneal epithelium in all rabbit experimentswas intact and covered with medical grade silicone oil. In the humancorneas, the epithelium was debrided prior to mounting in the specularmicroscope. At various times during the course of the perfusion, thecorneas were fixed for scanning and transmission electron microscopy in2.7% glutaraldehyde and phosphate buffer (pH 7.2, 330 mOsm) for at leasteight hours at 4° C. They were then post fixed in 2% osmium tetroxidefor one hour and embedded in a low viscosity epoxy medium. For scanningelectron microscopy (SEM), the resin was washed off the endothelialsurface of a portion of each cornea according to the modified method ofCleveland and Schneider, "A Simple Method Preserving Ocular Tissue forSEM," Vision Research 9, pp. 1401-1402, 1969. After polymerization ofthe tissue specimens overnight at 37° C. and for 48 hours at 60° C., thetissue was glued to SEM specimen stubs coated rotationally with carbonand gold palladium metal and viewed with an AMR (1000) scanning electronmicroscope. Endothelial perfusion of ophthalmic irrigating solution ofthe instant invention to the rabbit cornea resulted in a swelling rateof 0.001 μ/hr. On ultrastructural examination, the endothelial cells ofall corneas were intact with normal cell morphology maintainedthroughout the three-hour period.

In the paired corneas, the endothelium was perfused with Plasmalyte 148(pH 7.4). There was marked corneal swelling at a rate of 84.5 μ/hr. Uponultrastructural examination with SEM, the endothelial cells appeared tobe separating at the junctions between cells, and on transmission EM,the cells were balled up and the junctions broken. In some corneas thatwere perfused with Plasma-lyte, complete junctional breakdown occurredand the endothelial cells balled upon Descemet's membrane.

The paired corneas that were perfused with lactated Ringer's solutionhad a swelling rate of 32.7 μ/hr. On ultrastructural examination, thecells were morphologically swollen and there appeared to be breaks inthe outer plasma membrane. Cytoplasmic blebbing was apparent on theouter surface of the corneal endothelial cells and on transmissionelectron microscopy there was dilation of the endoplasmic reticulum,condensation of the mitochondria and microfilament network along theouter plasma membrane. Associated with the cytoplastic changes, therewas also edema of the endothelial cells adjacent to Descemet's membrane.

Endothelial perfusion of human corneas with ophthalmic irrigatingsolution of the instant invention had a negative swelling rate of 24.6μ/hr. and, upon ultrastructural examination, the endothelial cells wereshown to be intact, the junctions intact, and the cell organelles normalby transmission electron microscopy. The paired corneas that wereperfused with Plasmalyte all showed junctional breakdown between theindividual cells and balling of the endothelial cell. Theseultrastructural changes were associated with a corneal swelling of 19μ/hr.

By comparison, the paired corneas that were perfused with lactatedRinger's solution showed a slight deswelling of only -5 μ/hr. but therewere associated ultrastructural changes in the surface morphology of thecorneas of older humans. Junctional breakdown was apparent, and ballingof the cells and nucleus with marked vacuoles in the cytoplasm occurred.However, the cell organelles within the endothelium appeared normal.

The data obtained in this study indicates that Plasmalyte 148, anintervenous electrolyte solution used for phacoemulsification, causedcorneal endothelial cell breakdowns and functional disruption of theendothelium during in vitro perfusion and therefore may cause cornealedema after phacoemulsification. The data obtained also indicates thatthe ophthalmic irrigating solution of the instant invention maintainshuman and rabbit corneal function and ultrastructural integrity for athree-hour period.

By comparison, lactated Ringer's solution is only able to maintain thecorneal endothelial cell integrity and ultrastructural appearance tovariable degrees both in rabbits and in humans and may be adequate forshort-term irrigation of the cornea, but will provide, at the very best,minimal protection to the corneal endothelium when perfused to theendothelium for extended periods of time.

While the whole invention has been described in terms of a preferredembodiment, modifications obvious to one skilled in the art may be madewithout departing from the scope of the present invention which islimited only by the following claims:

What is claimed is:
 1. A method of maintaining corneal stability duringocular surgery comprisingpreparing a stable basic solution containingbicarbonate ions, sterilizing said basic solution and prepackaging saidbasic solution, preparing a stable acidic solution containing calciumions, magnesium ions, dextrose and glutathione, sterilizing said acidicsolution and prepackaging said acidic solution, providing sodium ions inone of said solutions, providing potassium ions in one of said solutionsand providing chloride ions in one of said solutions, mixing saidprepackaged solutions together in a manner which maintains theirsterility to form a combined solution containing between about 130 andabout 180 mM/l sodium ions, between about 3 and about 10 mM/l potassiumions, between about 1 and about 5 mM/l calcium ions, between about 0.5and about 4 mM/l magnesium ions, between about 10 and about 50 mM/lbicarbonate ions, between about 2 and about 10 mM/l dextrose and betweenabout 0.03 and about 0.5 mM/l oxidized glutathione or the equivalentamount of reduced glutathione, said combined solution having a pH ofbetween about 6.8 and about 8.0 and an osmolality of between 250 andabout 350 mOsm/kg, and within about 24 hours of mixing said prepackagedsolutions, irrigating an eye with said combined solution.
 2. A methodaccording to claim 1 wherein phosphate ions are provided in said basicsolution in sufficient quantity so that the phosphate ion concentrationin said combined solution is between about 1 and about 5 mM/l.
 3. Amethod according to claim 1 wherein said sodium ions are provided insaid basic solution.
 4. A method according to claim 1 wherein saidpotassium ions are provided in said basic solution.
 5. A methodaccording to claim 2 wherein sodium chloride, potassium chloride,dibasic sodium phosphate, and sodium bicarbonate are dissolved in waterto make said basic solution, and wherein calcium chloride, magnesiumchloride, dextrose and oxidized glutathione are dissolved in water tomake said acidic solution.
 6. A method according to claim 2 wherein saidcombined solution has a pH of between about 7.2 and about 7.8.
 7. Amethod according to claim 2 wherein said acidic solution has a pH belowabout
 5. 8. A method according to claim 2 wherein said basic solution isprepared by dissolving said phosphate prior to dissolving saidbicarbonate so that said bicarbonate is introduced into a solution at apH of above about
 8. 9. A method according to claim 2 wherein said basicsolution and said acidic solution are mixed at a volume ratio of basicsolution to acidic solution of between about 10:1 and about 40:1.
 10. Amethod according to claim 1 wherein said combined solution has anosmolality of between about 290 and about 320 mOsm/kg.
 11. A methodaccording to claim 1 said combined solution having a pH of between about7.2 and about 7.8.
 12. A method according to claim 2 wherein said sodiumions are provided in said basic solution.
 13. A method according toclaim 2 wherein said potassium ions are provided in said basic solution.14. A method according to claim 5 wherein said combined solution has apH of between about 7.2 and about 7.8.
 15. A method according to claim 5wherein said acidic solution has a pH below about
 5. 16. A methodaccording to claim 5 wherein said basic solution is prepared bydissolving said phosphate prior to dissolving said bicarbonate so thatsaid bicarbonate is introduced into a solution at a pH of above about 8.17. A method according to claim 5 wherein said basic solution and saidacidic solution are mixed at a volume ratio of basic solution to acidicsolution of between about 10:1 and about 40:1.